nm No. 24 
FILE Noxxxl-17 
COPY No. 
SECRET 
DUTCH SCIENTIFIC 
INSTITUTIONS IN UTRECHT 
AND AMSTERDAM 
SECRET 
COMBINED INTELLIGENCE OBJECTIVES 
SUB - COMMITTEE SECRET 
DUTCH SCIENTIFIC INSTITUTIONS 
IN UTRECHT AND AMSTERDAM 
MOULD METABOLIC PRODUCTS: 
MISCELLANEOUS SUBJECTS: 
Reported by 
Lt. Col. H.J. BARBER, M. of S. 
CIOS Target Numbers 24/28,24/28a, 
24/28b,24/98 & 24/199 
Medloal 
June 6,' 1945 
COMBINED INTELLIGENCE OBJECTIVES SUB-COMMITTEE 
G-2 Division, SHAEF (Rear) APO 413 3R0RST 
tahia: of so:ttskts 
Subject Page 
Part I Introductory and General observations, . 1 
Part II Interview with Professor KOningsborger. 
Status of Organisation 2 
Summary end Recommendations 2 
Introductoiy ;emorI:s ......... 2 
Information Available to KOningsberger 3 
Description of KOningsbergerfs work . 4 
Discussion 6 
Fart III Interview with Professor Jansen 
Status of Organisation * 8 
Summary and Recommendations 8 
General Remarks 8 
Detail of Isolation of Antibiotic 
Substances 9 
Part TV Interview with Dr. Timmerman 
Status of Organisation 12 
Summary and Recommendations 12 
Interview 12 
Part Y Interview with Professor KOgl 
Status of Organisation 14 
Summary and Recommendations '14 
Interview 14 
Investigator 
Lt. Col, H.J,Barber, 
Ministry of Supply Part I. Introductory and General'Observations 
The assessorsr preliminary observations and the 
investigators further examination of this of 
University Institutions makes it clear that they 
cannot really be regarded as O.I.O.S. targets in the 
sense -that they can provide useful information of imme- 
diate practical value. These Dutch University Insti- 
tutions have in general remained undisturbed by the 
Germans and have managed to continue with some of 
their scientific work under considerable difficulty. 
Dutch University life in the teaching sense has been 
practically dead for the past few years and many of 
the research workers have disappeared for various and 
obvious reasons. It could not be expected therefore • 
that any far reaching developments would arise in such 
circumstances and this is in fact the case. 
The .investigator takes the view- that these Dutch 
Scientific men have performed a valuable service to 
their country (and therefore to the .Allies as a whole) 
by maintaining some kind of continuity of work and 
preserving their integrity. They can and must play an 
important part in the full restoration of university 
life in every sense. The importance of this - especi- 
ally in such fields as Health Services - needs no 
emphasi’s end British and American authorities must 
make every effort to supply their urgent and essential 
needs s> that they can stand on their own feet as soon 
as possible. This question is probably dealt with in 
other places but it cannot be kept out of an account 
of these visits. 
The urgent needs are; 
1. Supplies of essential chemicals, biological 
standards, equipmon tetc, 
2. Close contact with allied.scientific men, 
3. Scientific publications (especially review 
journals) from 1940 onwards. 
This investigator would urge that this matter be * 
made the subject of special consideration as soon as 
possible by a competent body set up for the purpose. Part II, Interview with Or, V.J* Kflnlngsberger 
Status of Organisation 
Dr* KBningsberger ia Director and Professor of 
Botany at the Botanisoh Laboratorium, Utrecht. 
This institution is part of the Faculty of 
Soienoe of the University and works in close asso- 
ciation with the Pasteur Inatttuut. The Botanical 
gardens and buildings for research and teaching are 
situated at the rear of Professor ESningsberger1s 
residence, Nleuwe Gracht, 18 f, Utrecht*. 
Summary and Recommendations 
1. The work reported here has no immediate bearing 
on Penicillin production. 
2* Penicillium expansum produces "Expansin** (Patulinll 
3» These extracts have been assayed usiing Pythium 
mammilla turn and P0 de Baryanum. 
4. The therapeutic properties have been explored 
tentatively in lupus, tricho and epldormophy- 
totic infections.of the skin with some success. 
5. All work should be considered in relation to 
British work on Patulin. 
A varfeit of Professor KBningsberger to London to 
contact Professor Haistrick and other M. of S, 
Scientific advisers is desirable In the near 
future. 
7, This group of workers would be a suitable agency 
through which to establish Penicillin production 
in Holland. 
8, Van Luyk is examining oral flora in relation to 
head colds and infections of the upper respira- 
tory tract. 
Introductory* Remarks 
C.I.0,3, Secretariat, G.A.i-’.T, and "TM Force 
were all emphatic that Professor KBningsberger was 
a loading member of the Dutch Resistance movement and was therefore to be regarded as a friend. The 
interview was conducted in the light of these 
directions end the investigator was convinced that 
Professor KBningsberger was only too anxious to 
communicate all his information to allied scientists, 
although he was 'fully conscious of the fact that he 
was much behind other workers, largely owing to the 
difficulties under which he has worked and is still 
working. Professor KOningsberger Is in the course 
of translating a full report by Van Luylc on his 
work and this will be sent by him to "T? Force in 
the course of the next two or three weeks. This 
report is therefore confined to an assessment of the 
status of this work in relation to that of British 
and American Groups, It should also be born in 
mind that the investigator is an organic chemist 
whose knowledge of antibiotics is primarily chemical 
whereas these Dutch workers are microbiologists. 
Information Available to Professor 
Professor KOningsberger had obtained some in- 
formation on Penicillin from a leaflet dropped by 
the H.A.F, This was not of a detailed technical 
kind but was nevertheless of interest to him. 
Further details recently reached him in a photo 
copy of a paper from the Schwelzer I.iedizinische 
Wochenschrift June 1944, which reviews the infor- 
mation on antibiotics. This paper is probably 
available in England but part of a taale in /the 
paper is reproduced here as it illustrates the 
situation. 
Panicillia 
P, notatum Fleming Penicillin ~%£^2^ic 
P, notatum Westllng 
P, glauoum ' Penatin 
P. petulu.i • - Patulin 
P. claviforme 'Claviformin CgllgOg 
P, puberulum Bainier Panicillic GqII^oO/j. 
acid 
P, cyclopurin -do- -do- P, spinosum Thom. Spinulosin CqHq05 
(Toiu^inone 
derivative) 
P, citrinum Gitrinin c15^14^3 
P* resticulosum Peptide 
P, fimbriatum Gliotoxin" C14HX6K2S2°4 
Asnerglller 
A, flavus 
A, clavatus 
A., fumigatus Fresenius . Fumigatin G8H7°4 
A. fumigatus Fresenius 
A, fumigatus nut. Kelvoea Kevolic C32Ii4O0 
acid * ° 
A„ niger 
A gigentus Gigantic 
acid 
f 
The inaccuracy and incompleteness of the data 
on Penicillin is readily appreciated. This 
investigator has had access to the secret reports 
of the British G,P,3* Committee and the correspond- 
ing American documents and great care was taken 
during the discussions not to disclose any infor- 
mation not published. It was possible however, to 
inform Professor KiJningsberger on many points on 
which there has publication in England or the 
U. ,A# 
Description of Professor. KOningsberger's Work 
Culture 
The organism used was almost exclusively 
PenioilHum expa-sum. This was very easily ob- 
tained from a piece o£^apple in contact with 
ordinary soil in a Petri dish. The apple rapidly becomes covered with the mould and is removed to 
a sterile dish. The pure organism is readily 
obtained sin.ee, when grown in this way, it forms 
well marked upright core mi a which resemble macro- 
scopic stalks. The clean sep-ration of the-spore 
head is thus a simple matter. The culture medium 
is ICniJps solution with 2f'v sucrose added. It' is 
grown‘for 10 days at ;350G, If kept longer it 
gradually loses its power to produce the anti- 
biotic principle, nn obvious sign of this is the 
lightening in colour of the mould, an active one 
is dull green the inactive dirty white. 
Solutions of maximum antibiotic activity are obtained 
in 3 weeks and no culturing is done from such solu- 
tions. 
Professor KSningsborgsr stressed the need for • 
frequent re-isolation of Pi expansum from soil. The, 
isolated organism grown in pure culture media • 
loses its- activity in 3 months. I. notatun was 
stated to behove similarly but not P. claviformum. 
The investigator does not know if this agrees with 
British experience, kfiningsberger said, that the 
Germans obtained P. notatum from' the Bureau of 
Cultures at Caarn, but Van Luyk was confident that, 
as these had been grown for £5 years on pure culture 
media, they would have negligible power of producing 
penicillin. Some two or three ‘dozen Gorman insti- 
tutes had obtained str.-ins from Baarn, 
i 
Testing of Activity 
Professor KiJnihgsberger stressed that this 
method was*only an improvisation forced on them by 
the inadequacy of their recources, Pythiu.. 
mamnillaturi and Pythium de .Baryarium-su re used as 
test organisms. The technique is as fellows. 
Small rectangular sections (about 6x4 mm.) are 
cut with a punch from a sheet of agar inoculated 
with pythium spores and dropped into’ a tube con- 
taining tap water (control) or tar; wafer and the / 
ann-ropriate volume of the expansin liquor. The 
control rapidly develops a fibrous growth of pythium 
resembling very loose cotton and the height, to 
which this extends is clearly visible by transmitted 
light. In the control this usually extends about half way up the liquid. By serial dilution it is 
a simple matter to establish the concentration of 
active liquor v/aich just inhibits the growth of 
pythium. 
Isolation of active principles 
This has been carried out at Amsterdam and 
the methods employed are descrjbed in the report 
on the visit to Professor Jansen. It should be 
said here that there is every probability that the 
main product is Patulin but other substances may be 
produced. The plotting of “bacteriological spectra” 
would doubtless clear up many points of this kind. 
Discussion 
It is clear that this work has not reached a 
stage of immediate practical importance although 
future developments may well prove interesting. 
Progress is in the first place dependent on the 
restoration of the essential services - gas and 
electricity - needed for the running of thermo- 
stats, sterilising ovens, air conditioning., 
refrigerators and so on. It will,then be possible 
for Professor KSningsberger to assess his products 
by standard methods developed by allied scientists. 
He is aware of the Oxford Unit for Penicillin but 
he will obviously need sonle standardised Penicillin 
of a fairly high activity in order to establish his 
own technique. Sufficient should be obtainable 
from military sources in the locality in the first 
place (the amount would be extremely small) but 
later a very small Quantity of pure crystalline 
sodium pencillin II should be made available. 
KBningsberger will then be able to describe his 
products in knovm units and their true place in the 
scale of activities can then be seen. 
The investigator felt strongly that Kflnings- 
berger and Van Luyk may be a ole to. make very valu- 
able contributions in the field of antibiotics 
from various angles. They are two experienced men 
who have shown great ingenuity in surmounting 
difficulties, they have the space in good labora- 
tories in a fine modern building, they have the 
equipment and they have ideas. Van Luyk’s examina- 
tion of oral flora and"their possible bearing on 
infections of the respiratory tract referred to in 
Anderson’s assessment report is an example, as is 
also his early association with ideas on antibiotics. Ways and means should he found to integrate this 
work ith that of allied groups especially of 
Raistrick, Fleming and others. It is assumed th:.t 
the II. of S. will wish Penicillin production to be 
established in allied countries, and this group of 
Dutch workers in the opinion of the investigator 
would be a valuable nucleus on which to base a 
suitable set-up. 
It should not be difficult to work out a scheme 
of collaboration on lines similar to Anglo-American 
Co-operation on Penicillin in its various aspects. 
If this view is accepted in principle the first step 
should be to organise a visit of these Dutch geion- 
tists - particularly KSningsberger and Jansen to 
London to establish direct contact with the Ministry 
end its Scientific Advisers in this field. 
It should be added that this work in Holland 
has received some support from Dutch Chemical 
industry. The firm of Brocades, Stheeman and 
Phamaoia (Brocapharm) of Amsterdam is making a 
small annual grant believed to be equivalent to 
about £600. They have * as yet done nothing towards 
Semi-Scale development of the methods of preparation. 
They might have done had communications been 
possible. Enquiries in England show that this firm 
is the leading house of its kind in Holland and 
enjoys a good reputation. 
The complete paralysis of all communications in 
Holland has to be experienced before it can be 
appreciated. There are no trains,' no buses, no 
trams, no postal service, into or in Holland (apart 
from B.L.A.), Professor KOningsberger in Utrecht 
oannot keep in touch with Professor Jansen in 
Amsterdam, There are many bicycles but most have 
to be ridden without tyres. Wood blocks have been 
removed frQ*i roads for fuel. Utrecht has no gas or 
electricity and a reasonable water supply has only 
just been achieved. This parenthetical note has 
bean Inserted to emphasise the difficulties facing 
these scientists at present, ‘ They are, however, 
cheerful and uncomplaining to a remarkable degree 
and are planning energetically for the restoration 
of university life in Holland. 
Date of Visit: 26th May, 1945. Part III. Interview with Professor 3.G.P, Jansen 
Status of Organisation 
r ■ " ‘ 1 11 
Dr. Jansen is Director of the Physiological 
Chemistry Laboratory at the Netherlands Institute 
of nutrition. 
J.D, Meyerplein, 3, .Amsterdam, 
This laboratory is a department of the 
University of Amsterdam and much of its work is 
on problems of nutrition including vitamin assays 
and C) on much of Holland’s food produce. 
Summary and Recommendations 
1, This report should be read in conjunction with 
that on Professor KBningsberg-r’c work at 
the Botanisch Laborstorium Utrecht. 
2, The main product from the growth of Penicillium 
expansum is almost certainly Patulm, The 
Dutch workers have called it Hxpansin. 
3, Growth of P. expansum at low temperature may 
produce a new antibiotic with high activity 
against S. aureus. 
General Remarks 
Professor Jansen took the investigator from 
his house to the laboratories and showed him round 
them. These were old and had obviously suffered 
from neglect during the war years. Many of the 
staff had perforce to retire into hiding and were 
now slowly returning to work in the university. 
The laboratories were equipped for semi-scale 
operations though not extensively so, Jansen had 
not received any culture fluid from K&ningsberger 
in Utrecht for many months owing to lack of communi- 
cations and in short he had made very little pro- 
gress in the last few months, 
’The laboratory was concerned with nutritional 
problems and conducted Vitamin B- and G assays. They 
had a small rat house for biological assays and they 
had the usual physical apparatus. The photoelectric 
colourimeter they used for assays they had built 
themselves. A Hilger Spekker had recently been taken out of its hiding place during Genian 
occupation. 
Detail of Isolation of Antibiotic Substances 
Expensin 
The original Method of isolation was as 
follows. The culture fluid was concentrated at 
low (sic.) temperature under reduced pressure, 
Jansen estimated the temper, ture to be a..out 40 0o 
and said the liquor was about'neutral, The inves- 
tigator is confident that no extra special pre- 
cautions were taken to prevent the destruction of 
very labile substances w: 1oh night be present. The 
acidified concentrated*liquor was then continuously 
extracted 'with ether. The continuous extractors 
were quite normal in design and hold two or three 
litres of aqueous solution, A battery of about 
6 of these was set up in c small downstairs room 
devoted to this kind of d,.oration with* inflammable’ 
liquids. The eth r solution was trc.ted with con- 
centrated hydrochloric add and this drew cut a 
The run was extracted with boiling benzene 
and the benzene solution, sometimes without further 
concentration, deposited the crystalline product 
tfhey cell "Expanrln". Sometimes it was necessary 
to pass the* bonzone solution through a coluom of 
alumina in order to obtain it in crystalline condi- 
tion. The m.p. quoted by Jansen was ICC0 and the 
m.p. .of patulln is 112° so that there is every 
probability that this product is in fact patulln, 
Jansen had isolated a product from panic llliusi 
pstulinum and found it identical. What has not 
been done is to confirm by mixed m.p. the identity 
of the Dutch Dxpanoin with the British Patulln, It 
is just possible tint P, patulinum of diiferont 
ori.rjin in different nutrient -media might produce a , 
modified Patulin. Jansen’s -Bxpansin has s very 
high activity against Pythium,mammillaturn 
relatively low activity agvnnst S. aureus. The 
figures -are; 
Pythium mammillaturn 1 - 5,C0C,0C0 
Staph. Aureus 1 - 50,UUC 
Other Ar tieiot ics 
Later Jansen improved the method of isol- tion 
on the following lines. The culture'fluid was 
passed through a norite column, vmich adsorbed all 
the active principles. An important point to note is that, unless the norite column is pre-treated with 
seme substance which partly saturates the high 
adsorptive power of the charcoal, the activity can- 
not be recovered from the charcoala Iqueous solu- 
tions of benzoic acid were passed through the column 
first. The charcoal was extracted with methanol in 
the first place but later it was found that ether 
was better. The ether solution on evaporation and 
allowing to crystallise yielded ejepansin as before 
but the liquors contained another substance which 
has not been isolated in a pure condition* This has 
a higher activity against S, aureus and a lower 
activity against P. mammillatum, Jansen is confi- 
dent that is another very active substance 
present. 
„A further observation of some interest was made 
fortuitously. Some liquor from KJJningsberger, at 
Utrecht was delayed for some months in transit and 
was then found to contain little or no expensin'. 
It did however contain material which had a high 
activity afeainst S. aureus. This hypothetical, sub- 
stance must be a somewhat stable one. It may be the 
same as that responsible for the activity referred 
to in the preceding paragraph. 
Yet another observation has been made. If 
the culture is rapidly stirred or aerated there is • 
produced a fairly high S. aureus activity moderately 
quickly but after reaching a peak this rapidly 
disappears. However if the P. expansum is grown at 
low temperature 5-10° then little or no expansin 
is produced, but a high S. aureus activity results. 
Additional evidence for the existence of this 
principle was quoted by Jansen. They have studied 
the anti-urease activity of various fractions and 
have found that solutions containing expansin have 
low activity but solutions freffa which expansin has 
been removed or in which it has not -been produced 
have a high activity. Clearly there are some 
features of interest here. 
Janseiq hopes to publish these results shortly 
in the Arphiv/der Physiologic. Hollandaise. 
Jansen.was very co-operative and helpful (he 
has a son in the Dutch Army in^England who is 
qualifying in medicine). Again the remarks made in the report on Professor KOnin^sber^er1s work 
apply here. Means should bo found to re-establish 
contact with the scientific world, Jansen counted 
himself lucky in having a copy of the Annual 
Meview on Diochemistry' for 1940. This must be 
remedied. 
Date of Visit: 26th May, 1945. Part IV . I n t e rv ie v; v ■ i t h Dr, -Timmerman 
Status of Organisation 
Dr, Timmerman is Director of the Pasteur 
In- tituut and :.yks Instituut voor de V'olksgeoendheid, 
Utrecht. 
SurgQ- ry eng reconiricndations 
1. The Institute has been engaged almost entirely 
on the production of essential sera and 
vaccines and research work has been very 
limited. ■ 
£„ It 'is inportcnt for the Health Services of the 
. province that this Institute be restored to 
full efficiency as soon as possible. 
3. The needs quoted on p.l. of this report are 
particularly important to tliis Institute. 
Interview 
This target was visited by this investigator 
at the reouest of C,A,f,T. Group. 3,, as it was close 
to the primary target. It should be noted that the 
investigator is an Organic Chemist and the work of 
the Institute is Biological. There is little to 
add to the Assessors* report on the activities of 
this Institute and-Dr. Timmerman made it quite clear' 
that research v/ork had been almost out of the ques- 
tion durin the occupation period. There is there- 
fore no information of immediate value to C.3.0.S, 
The Institute and its Director has a high scientific 
st tus and its work obviously has great importance 
to the Health Services of Utrecht and the surrounding 
'rea. Clearly evsry effort should be made to restore 
the Institute to full efficiency-. Apart from 
essential services, such as gas and electricity, 
v;hich are not the concern of this investigator 
cert-in International Biological Standards are 
urgently required. These are; 
1„ Pituitary Posterior Lobe. 
2, Insulin. 
3. Su,lpharsphenamine. 
4 • Neo-Arsphenamine . 
5, An^urin. Dr, Timmerman has already attempted to communi- 
cate with Sir Percival Hartley of the National 
Institute for Medical Research for these standards 
but doe? no'f •'qw the result. The investigator 
promised he would e:p edite this matter and 
has been able to arrange with Sir Percival Hartley 
and the C.I.O.S, Secretariat for the rapid dispatch 
and conveyance of these urgently needed materials. 
This target, being, quite distinct from an 
enemy one, the investigator was able to answer 
questions on the whereabouts and welfare of Dr, 
Timmerman’s numerous British friends in Scientific 
circles. It is difficult to convey in sufficiently 
graphic terms the sense of isolation from outside 
scientific circles that these Dutch Scientists have 
experienced for so long. This can only be remedied, 
slowly, but the most effective way of doing this 
would be to grant facilities at an early date for 
leading scientific men such as Dr, Timmerman to 
visit London and other Allied centres to re-establish 
personal contact with British and American scientific 
thought. 
Allied Scientific journals have naturally 
not reached Holland and the sup. lv of these aunt 
have a 4*igh priority when the first essentials h .ve 
been dealt withi Such journals as British • Journal 
of Pharmacology and Experimental Biology, Journal 
of Pathology and Bacteriology, end Physiological 
Reviews would be of considerable help. 
The investigator appreciates that this report 
stresses what we should do to assist Jr. Timmerman 
end indicates that he con supply no help to us. 
But it must be realised thatDr, Timmerman and many 
others h<*ve contributed much to.the Allied cause 
in the past years by their resistance ¥to German 
penetration and by maintaining under extraordinary 
difficulties the coherence ahd .integrity of the 
institution. 
Dr. Timnirman expressed his deep appreciation 
of the visit of the investigator. 
Date of Visit: 25th May, 1945. Part Y. Interview with Professor KBgl 
- Status of Organisation 
Dr, F, KB_1 is Director of the Laboratory of 
Organic Chemistry'. This is a part of the university 
and is endowed by the Rockefeller Foundation and 
also by the Dutch Government. It is situated in 
modern buildings (teaching and research) in 
Croesesstraat, 50, Utrecht. 
Summary and Pec omr.ier.da t ions 
1, KBgl claims to have substantiated"his observa- 
tions (subsequently disputed by other workers) 
that d-glutamic acid and not the normally 
occurring 1' isomer is produced in malignant 
tissue, 
0, This is produced by a different mechanism from 
that which synthesises the 1 isomer. 
3, £Bgl has shown that his ( /->;) biotin differs 
slightly fro the ( biotin of du Vigneaud. 
4, Hone of these matters is of immediate practical 
application. 
5, It is desirable that KBgl should contact allied 
scientists in London in the fairly near future. 
Interview 
There is little of material importance to add 
to the outline given by th.- assessors, professor 
KBgl discussed his results in detail quite freely 
with this investigator, It was obvious that KBgl 
was very unhappy about the doubt which had been cast 
upon his results by the failure of American workers 
(published) to repeat his findings. They had again 
confirmed them at Utrecht and the workers at Munich 
under Hans Fisher had also been able to confirm 
these findings. KBgl said that work using heavy water 
fed to his animals had enabled him to prove that the 
d-glutamic acid was formed by a different mechanism 
and was not produced by race..iisation followed by preferential elimination of the 1 isomer with con- 
squent increase in the proportion of the d, 
K5gl thought this difference between the behaviour 
of normal and malignant tissue was of profound 
significance in cancer. The investigator is 
not. competent to express an opinion on this point, 
but it is very ddubtful if this factor could immedi- 
ately point the way to new approaches to the prob- 
lems of cancer research. In view of the fact that 
the work has been written up for publication the 
investigator pressed K5gl to send it with as little 
delay as possible to a British or American 
scientific journal. The investigator suggested 
Journal of Biological Chemistry as a possible 
medium. Later, when contact can be re-established 
between these Dutch scientists and their British 
and American colleagues, it seems highly desirable 
that K3gl should have the opportunity of vindicating 
himself by demonstrating the technique which has 
given these results. But this is a matter for a 
scientific society or academic institution. 
KOgl has also done some work on biotin and 
demonstrated that the biotin which he has isolated 
differs from the one whose constitution was eluci- 
dated by du Vigneaud. The difference is in the side 
chain as follows 
, / cn nt 
l du Vigneaud -CII0GH2CE2CH2COOE Kflgl -CE-CH. 
1 CH~ 
COOH 
It will be noted that the total number of carbon 
atoms is the but that the carboxyl group is 
the heterocyclic system and the chain is 
branched in II3glts 3 biotin. 
This work has been published (Hoppe-Seylerj 
281, 65 (1944) and should be available in England 
by now. 
Date of Visit: 25th May, 1945.